Jul 28, 2023

Public workspaceWestern blotting

 Forked from Western blotting to detect ATP13A2 and ATP13A3
DOI
dx.doi.org/10.17504/protocols.io.ewov1oemklr2/v1
  • 1KU Leuven
rosanne.wouters
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DOI: dx.doi.org/10.17504/protocols.io.ewov1oemklr2/v1
External link: https://doi.org/10.3390/biom13020337
Protocol Citation: Marine Houdou, rosanne.wouters, Peter Vangheluwe 2023. Western blotting. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1oemklr2/v1
Manuscript citation:
Houdou M, Jacobs N, Coene J, Azfar M, Vanhoutte R, Haute CVd, Eggermont J, Daniëls V, Verhelst SHL, Vangheluwe P, Novel Green Fluorescent Polyamines to Analyze ATP13A2 and ATP13A3 Activity in the Mammalian Polyamine Transport System. Biomolecules 13(2). doi: 10.3390/biom13020337
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 01, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 82745
Keywords: ASAPCRN
Funders Acknowledgement:
ASAP
Abstract
Protocol to detect proteins via Western Blotting.
Materials

  • Antibodies:

- Goat anti-mouse IgG (H+L) secondary antibody HRP conjugated: Thermo Scientific, 31430
- Goat anti-rabbit IgG (H+L) secondary antibody HRP conjugated: Thermo Scientific, 31460
- Mouse monoclonal anti-GAPDH (lot #067M4785V, dilution 1:5,000): Sigma, G8795.
- Rabbit anti-ATP13A2 antibodies (lot #0000102992, dilution 1:1,000): Sigma, A3361.
- Rabbit anti-ATP13A3 antibody (lot # 000035781, dilution 1:2,000): Atlas Antibodies, HPA029471.

  • 0.25% Trypsin-EDTA: Gibco, 25200056

  • Dulbecco’s Phosphate Buffered saline modified without calcium chloride and magnesium chloride (DPBS): Gibco, D8537

  • Micro-BCA Protein Assay Kit: Pierce BCA Protein Assay Kit, Thermo Scientific, 23225

  • NuPAGE LDS sample buffer: Invitrogen, NP0007

  • Ponceau staining: Sigma, P7170

  • Pre-cast 4-12% Bis-Tris gels: Invitrogen, NP0321BOX

  • PVDF membranes: Thermo Scientific, 88518

  • RIPA Lysis and Extraction Buffer: Invitrogen: 89900

  • SIGMAFAST Protease Inhibitor Cocktail Tablets, EDTA-Free: Sigma: S8830

  • SuperSignal West Pico PLUS chemiluminescent Substrate: Thermo Scientific, 34095
Harvesting cells
Harvesting cells
collect the cells by scrapping them with a scrapper in Dulbecco’s Phosphate Buffered saline modified without calcium chloride and magnesium chloride (DPBS) or, using 0.25% Trypsin-EDTA for which stop enzymatic reaction by adding culture medium.
Centrifuge cell suspensions at 450 g, 4°C for Duration00:05:00 .

5m
Resuspend cell pellets with DPBS and centrifuge following the same indications as in 2. Repeat once.
Discard supernatants and keep cell pellets on ice.
Cell lysis and protein concentration determination
Cell lysis and protein concentration determination
Resuspend cell pellets in RIPA buffer (RIPA Lysis and Extraction Buffer supplemented with protease cocktail inhibitors.
Vortex Duration00:00:30 and keep on ice for Duration00:30:00 .

30m 30s
Centrifuge at 20,000g, 4°C for Duration00:30:00 .

30m
Keep supernatants on ice to proceed with protein concentration determination using the micro-BCA Protein Assay Kit.
SDS-PAGE
SDS-PAGE
7h 10m
7h 10m
Loading
Mix 20 µg of protein with NuPAGE LDS sample buffer and 5% β-mercaptoethanol final.
Load protein on pre-cast 4-12% Bis-Tris gels. Include at least one lane with a protein ladder.
Running
Run for Duration00:10:00 at 100V and Duration01:30:00 at 110-130V.

1h 40m
Transfer
Transfer onto PVDF membranes using a liquid transfer and following settings: 100V, Duration01:15:00 , 4°C.

1h 15m
Scan membrane if necessary.
Blocking
Duration01:00:00 Block membranes with blocking buffer (5% milk powder in 1X TBS and 0.1% Tween20 (REF)) for Duration01:00:00 at room temperature, 19 rpm.

2h
Primary antibodies
Incubate membrane with primary antibodies in solution (1% bovine serum albumin in 1X TBS-Tween20 (TBS-T) buffer), DurationOvernight at 4°C, 19 rpm.

1h
Wash membrane three times for Duration00:05:00 in TBS-T, 19 rpm.

5m
Secondary antibodies
Incubate membrane with peroxidase-conjugated secondary antibodies in solution (1% milk powder in 1X TBS-T) for Duration01:00:00 at room temperature and, 19 rpm.

1h
Wash membrane five times forDuration00:05:00 in TBS-T, 19 rpm.

5m
Detection
Use a chemiluminescence reagent to detect signal and acquire with a Biorad Camera (Vilber Lourmat) and its software (ImageLab).